To understand better the specificity of peptide binding by MHC class I molecules, we have evaluated the capacity of a panel of unrelated peptides to compete for the presentation of viral peptides presented by HLA-A2, HLA-A3, HLA-B27, and HLA-B37. Out of 41 peptides tested, only five bound to more than one of the MHC molecules analyzed. Pairwise comparisons of the peptide binding specificities among these four different class I molecules revealed no common competitor peptides in four of the six possible comparisons. Thus, each class I molecule appears to have a functionally distinct peptide binding site, as reflected by the ability to bind largely non-overlapping sets of peptides. In understanding CTL anti-viral responses and in creating vaccines designed to elicit CTL responses, it is critical to identify the portions of viral proteins that bind class I molecules and that are recognized by T cell receptors. Previous findings have indicated that a significant portion of the CTL response of H-2d mice to influenza virus is specific for one of the viral polymerases (PB2). To identify the region of PB2 naturally processed and presented by influenza virus-infected mouse cells to CTL, 31 PB2 peptides of 9-16 residues in length were chosen and chemically synthesized. Two peptides, PB2, residues 146-159 and 187-195, were found to sensitize histocompatible target cells for recognition by influenza virus-specific CTL. When CTL were generated to individual viral proteins using influenza-vaccina recombinant viruses, we found, to our surprise, that PB2-specific CTL failed to recognize cells sensitized with PB2 peptides 146-159 and 187-195. Further analysis showed that these PB2 peptides were, in fact, recognized by nucleoprotein (NP)-specific CTL generated by NP-vac virus priming and influenza A virus stimulation, or NP peptide stimulation in vitro of NP-vac or influenza A-primed CTL. These results demonstrate that when screening peptide libraries one cannot assume that positive peptides necessarily identify the viral protein to which the CTL response is directed.